1. Technical Field
This invention relates to a biochip comprising several molecular recognition areas and a device for reading such a biochip. A biochip means any chip or support with one or several areas (called recognition areas) on its surface, equipped with molecules with recognition properties. Throughout the rest of this text, the term biochip is used improperly, independently of whether the chip is used for a chemical or a biological analysis.
For example, recognition molecules may be trace nucleotides, polynucleotides, proteins such as anti-bodies or peptides, lectines or any other ligand-receptor type system. In particular, recognition molecules may include fragments of DNA or RNA.
When the biochip is brought into contact with a sample to be analysed, recognition molecules can interact, for example by complexing or by hybridisation with xe2x80x9ctarget moleculesxe2x80x9d of the sample. Thus, by equipping a biochip with several recognition areas with different recognition molecules selectively sensitive to different target molecules, it is possible to detect and possibly quantify a large variety of molecules contained in the sample. Each recognition area contains only one type of identical molecules.
Complexes formed on the biochip may be identified by means of fluorescent marking applied to target molecules of the sample.
The read device according to the invention is intended to facilitate the operation to read marked or unmarked molecules that may be present in chip recognition areas.
Recognition areas may be read without the presence of any marker, and this type of technology is already known in the state of the art. In particular, some direct methods of detecting hybridisation include detection of a variation of the mass, a variation of the thickness, and a variation of the index. Photothermal methods are also known, and are described in document 1 that is mentioned in the references at the end of this description. Finally, Boccara et al. have described a photothermal deflection technique in documents 2 and 3. Improvements to this technique were then described in document 4. The references of these documents are given at the end of the description.
Thus, the invention is used for applications in the biological and chemical analysis fields.
Particular applications in the biological analysis field may include the search for polymorphisms and mutations, sequencing by hybridisation and monitoring the expression of genes.
2. State of Prior Art
The number of recognition areas in a chip varies depending on the type of analysis to be carried out. Thus, a distinction is made between xe2x80x9clow-densityxe2x80x9d chips that comprise a few tens to a few hundreds of recognition areas, and xe2x80x9chigh-densityxe2x80x9d chips that may comprise several thousand or several hundred thousand areas of this type.
The size of recognition areas on high-density chips is small. The dimensions of these areas are less than 100 xcexcm, or possibly even less than 10 xcexcm.
As mentioned above, complexes formed on biochips are marked using fluorescent markers. For example, markers such as fluoresceine or phycoerythrine may be coupled directly on target molecules of the sample to be analysed. Target molecules may also be marked by means of indirect recognition groups such as biotin or digoxigenin.
Thus, when recognition molecules for a given recognition area have interacted or hybridised with marked target molecules, the fluorescent marker is fixed on these areas.
Reading a biochip includes excitation of fluorescent markers under the effect of light called the excitation light directed at the chip, and then recording of the fluorescence caused by the excitation light for each recognition area.
Detection of fluorescence in a recognition area enables a decision about the presence of target molecules (marked molecules) that could interact with the known recognition molecule, in the sample to be analysed, knowing the type of recognition molecule present in this area. The intensity of the fluorescence may possibly be measured to deduce the concentration of the target molecules concerned in the sample.
The reader can refer to documents such as 6, 7 and 8 for examples of these techniques, particularly for genetic biology applications. The references of these documents are given at the end of this description.
For low-density biochips, recognition areas may be read with imagery stations equipped with charge coupled devices (CCD). These stations are not very well adapted to high-density chips. CCD cameras should have a larger number of detection pixels, and since fluorescence light fluxes are particularly low for high-density chips, the cameras also need to be cooled to improve their signal to noise ratio.
Thus, for high-density chips, fluorescence scanners are used to scan the chip so that the recognition areas can be analysed in sequence.
These scanners are provided with a confocal optical system associated with a photo-electric sensor, to record the fluorescence in each area. The scanner can be used to observe objects with a very good spatial resolution (from 1 to 10 xcexcm) and the confocal optical system is a means of overcoming parasite light emission effects (auto-fluorescence, specular reflection, etc.).
As an illustration of a scanner for high-density chips, the reader could refer to document 4, the reference of which is also given at the end of the description.
Fluorescence scanners output electrical signals that are acquired to form a two-dimensional image of the biochip. The signals are also used to recognise the spatial structure of the surface of the biochip and to identify and delimit the recognition areas located on it.
Finally, the intensity of the signal for each recognition area is recorded as the result of the analysis.
These analysis results can then be subjected to an appropriate computer processing to obtain biologically or chemically relevant information.
It is found that the signal processing mentioned above has the disadvantage that a larger number of measurement points, or pixels, are required for each recognition area.
A precise delimitation of each recognition area requires a sufficiently high pixel-image density for each recognition area.
In practice, it is found that the number of pixels needs to be of the order of 36 to 64 for each recognition area, depending on its size, to be able to perform signal processing under acceptable conditions.
Thus, signal processing for high-density biochips requires major computer data processing and storage means. Therefore, processing is expensive.
Furthermore, segmentation of the image based on recognition areas is not perfectly reliable.
Documents 9 and 10 mentioned in the references at the end of this description describe other possible means of reading a biochip that avoid some of the difficulties mentioned above.
Document 9 describes how to place recognition and marking elements on the chip to make reading easier, making it possible to envisage reading biochips using reading devices such as compact optical disk players (CD-ROM).
In document 9, the authors describe a chemical treatment for biochips to make them readable on a reading device distributed to the general public (and particularly CD-ROM compact optical disk players). Before analysing the sample containing target molecules to be analysed, molecular recognition areas on the biochip are covered with a reflecting film formed by metallic balls anchored to its surface by xe2x80x9cbridgexe2x80x9d molecules. These reflecting balls are functionalised to make them bond to target molecules in the sample. Therefore during hybridisation, the same target molecule needs to be bonded at two points; firstly to the surface of the biochip on molecular recognition areas, and secondly to the surface of one of the metallic balls located above this recognition area. After hybridisation, an appropriate chemical treatment breaks the bridge molecules anchoring the metallic balls to the surface, and the surface of the biochip is rinsed. Only the metallic balls retained at the surface by a minimum number of target molecules are present at this time, and form biological information xe2x80x9cbitsxe2x80x9d.
After the processing proposed in document 9, an additional biochemical step is necessary for the analysis. This step involves the cleavage of bridge molecules. Furthermore, hybridisation of target molecules on recognition areas is very much slowed down by the presence of metallic balls that considerably reduce the rate of diffusion of target molecules towards the surface supporting the recognition areas. Therefore, the analysis can be very slow. Furthermore, the relation existing between the number of metallic balls remaining finally bonded to the surface and the initial concentration of target molecules in the sample, cannot be easily quantified. It is more like a step type relation. (There is a threshold below which the balls are not bonded and above which they are bonded. The dynamic range within which there is an intermediate number of attached balls, and therefore a number that can be quantified, is probably very small).
According to document 10, the biochip is provided with marks associated with recognition areas. However, these marks are distinct from the recognition molecules and are physically separate from recognition areas. The marks may be detected independently of a hybridisation or complexing reaction when the biochip is scanned.
As soon as the position of the marks is known, the time elapsed between successive detection of two marks can be measured, to establish a function determining the relative position of the scanner on the biochip. This function can be used to more precisely determine the location of the recognition areas on the biochip.
The use of marks on the biochip is a permanent means of improving the positioning of measurement areas and thus facilitating reading.
However, the relative displacement between the biochip and/or the scanner optical reading system need to be controlled sufficiently accurately for precise guidance of the optical scanner system on the recognition areas.
This control is not particularly difficult when the recognition areas are sufficiently large and there are not many of them. The relative displacement between the biochip and the optical system may be made efficiently using relative inexpensive mechanical means.
However, for high-density chips for which the dimensions of the recognition areas do not exceed a few microns, extremely precise mechanical means are essential in order to achieve satisfactory scanning of recognition areas and to obtain a sharp and undeformed image.
Extremely precise mechanical means are also necessary to achieve scanning of small recognition areas at a regular speed so that the image obtained can be corrected, and so that the image can be used as a function of the displacement.
As an illustration, consider the example of a high-density chip comprising for example 300xc3x97300 adjacent recognition areas with a 20 xcexcm side, and for a typical 7xc3x977 points measurement sample, a displacement resolution of 3 xcexcm is necessary, the precision of this displacement must be 1 xcexcm (xc2x10.5 xcexcm) and the positioning repeatability must be 1 xcexcm (xc2x10.5 xcexcm). The displacement speed must be constant and the scanning displacements must be parallel within 0.3 mrad. Inexpensive mechanics cannot achieve these characteristics.
Furthermore, the chip has to be oriented in space and displaced with a resolution of 10 xcexcm, a precision of about 5 xcexcm and a repeatability of the order of 10 xcexcm, in order to focus the optical system before the chip is read. These characteristics are given for a 100 xcexcm section depth of the optical system. A reduction in the depth of the section to 10 xcexcm would then require a minimum resolution of 2.5 xcexcm, a precision of 0.5 xcexcm and a repeatability of 1 xcexcm, on the focussing.
The need for high precision mechanical means is why biochip reading devices are particularly expensive.
Furthermore, since high-density biochip recognition areas have a small surface area, the biochips have to be scanned slowly to be able to collect a sufficient quantity of light energy for each sample of each recognition area.
However, slow scanning makes the analysis of the biochip excessively long whenever there is a large number of recognition areas.
The light quantity produced by fluorescence may be increased slightly by exciting the target molecule markers by means of powerful lasers. However, the use of this type of equipment further increases the cost of read devices.
Documents 11, 12 and 13 also illustrate the state of the art. Document 11 relates to detection and quantification of cells, but not to molecular recognition. Documents 12 and 13 describe read systems using slow positioning mechanisms that do not cause reading problems xe2x80x9cin real timexe2x80x9d.
One purpose of this invention is to propose a biochip read device that does not have the difficulties mentioned above.
One purpose in particular is to propose such a device at low cost capable of reading high-density biochips.
Another purpose is to propose such a device capable of more quickly scanning chips, and thus reducing the analysis time without reducing the measurement quality.
Yet another purpose is to propose a device capable of quickly identifying the position and orientation of a recognition area without making an oversampled acquisition of the chip image.
Another purpose of the invention is to propose a biochip adapted to the said reading device in order to minimize the cost of scanning mechanisms.
In order to achieve these purposes, the objective of the invention is more precisely a device for reading a biochip comprising several recognition areas and several optical positioning marks, in real time, the recognition areas comprising fragments of DNA or RNA and occupying predetermined positions relative to the optical positioning marks, the device comprising:
an optical head capable of projecting incident light onto the biochip,
means of relative displacement between the head and the said biochip, capable of scanning over the biochip,
a first optical system called an analysis system associated with the said optical head to project light possibly coming from recognition areas, onto at least a first electro-optic sensor,
a second optical system called a positioning system associated with the optical head to project any light from at least one positioning mark, onto at least one second electro-optic sensor, and
means of servocontrolling the means of displacing the said optical head to control these displacement means as a function of electrical signals from the electro-optic sensor in the optical positioning system, in which:
the displacement means comprise macroscopic (large scale) displacement means and microscopic (small scale) displacement means and in which
the servocontrol means are connected to the microscopic displacement means.
In the rest of this text, the expression xe2x80x9clight from marked molecules . . . xe2x80x9d will be used for convenience to refer to light that may be emitted as fluorescent light, or reflected or diffused or refracted from recognition areas in response to incident light, for example by specific groups used on marked or unmarked molecules.
Microscopic displacements are used to refine the position of the optical head along at least one of the three axes. (A first axis corresponding to the optical axis of the first optical system, and two axes perpendicular to the first axis).
A recognition area means a portion of the biochip on the surface of which there are molecules that have the property by which a given type of target molecules can be recognized.
Servocontrolling scanning by the optical head to the positioning system electrical signals is a means of correcting the relative displacement between the chip and the optical head in real time, to obtain extremely precise positioning with inexpensive displacement mechanical means. In particular, it becomes possible to use mechanical means such as actuators that are usually installed on compact disk players.
Servocontrol is also made to achieve a displacement with a relatively uniform velocity to enable continuous reading of the fluorescence of recognition areas.
In this respect, it is worthwhile mentioning that the optical positioning system associated with a servocontrol means provides a means of reading the biochip in real time. In other words, it provides a means of knowing the read position with respect to recognition areas and/or the optical positioning marks, in real time.
Furthermore, the focussing precision obtained by the servocontrol enables the use of a confocal optical system with a shallow section depth so that the influence of parasite light originating from the biochip substrate can be reduced. Thus, better signal dynamics can be obtained.
Optical systems in the read device, unaffected by parasite light due to the low cross-section depth, may be made with a larger digital aperture and consequently collect more light. Consequently, faster reading is possible and/or the incident light source may be less powerful.
According to one aspect of the invention, the relative displacement means may include first macroscopic displacement means (large scale) and microscopic displacement means (small scale). In this case, as mentioned previously, the servocontrol means control the microscopic displacement means.
According to another advantageous aspect of the invention, the optical head may comprise a focussing lens and at least one axial lens focussing displacement actuator. A third optical system called the focussing system may be used with the optical head to project light due to reflection of incident light on the biochip onto a third electro-optic sensor, and focussing servocontrol means may be provided connected to the actuator to control the lens focussing displacement.
Due to the focussing servocontrolling means, recognition areas may be read continuously at the same time as focussing is being done. This characteristic also provides a means of not losing marking on the biochip, such that the measurement precision can be increased independently of scanning conditions.
According to one particular simplified embodiment of the invention, the device may comprise a unique optical system including the first and second optical systems and at least one electro-optic sensor common to the first and second optical systems, the said common optical sensor collecting light not only originating from molecular recognition areas, but also light originating from the positioning marks. The common sensor is then connected to a signal processing system and to means of servocontrolling scanning by the optical head.
Consequently, in this embodiment the common optical sensor outputs signals used for analysis of fluorescence and for servocontrolling the relative displacement between the chip and the optical head (scanning).
The invention also relates to a biochip comprising several recognition areas and several optical positioning marks.
The recognition areas according to the invention are wholly or partly superposed on optical positioning marks so as to cover all or some of the said marks.
For example, the optical positioning marks associated with recognition areas may comprise areas reflecting excitation light. They help to orient the biochip, identify locations of recognition areas, and make a precise scanning independently of whether or not hybridisation was done. In particular, optical positioning marks may be presented in the form of tracks called guide tracks.
According to one particular aspect, the positioning marks may be designed to have a specific reflectivity for incident light, different from the reflectivity of adjacent recognition areas.
According to another particular possible embodiment of the chip, a specific optical mark may be associated with each recognition area. Thus, there is no need to form an image of the entire biochip to determine the location of each area.
Thus, the possibility of precisely determining the location of recognition areas is a means of making precise measurements without oversampling, with a small number of pixels for each recognition area.
Furthermore, as soon as each recognition area is identified, a local analysis can be carried out for one or several given areas by moving the optical head or the chip, in order to aim the head so that it is facing the required area(s) directly.
There are several available possible means of recording light from chip recognition areas.
According to a first possibility, the optical head can be held motionless facing a recognition area and a signal acquisition can be made for a determined time before going onto the next area.
As already mentioned, the optical marks and the servocontrol means are used to position the head with sufficient precision so that a reliable analysis can be made. Furthermore, the optical marks are used to precisely position a recognition area even if it does not emit any fluorescent light.
Sensor signals can also be acquired without stopping, by displacing the optical head continuously over several recognition areas placed side by side such that the recognition areas cover the active surface of the biochip, for example by juxtaposition. In this case, the signals are integrated to determine the quantity of light received during the time necessary to scan one or several recognition areas.
However, fluctuations in the displacement speed may affect the analysis of the light quantity emitted per unit time by recognition areas.
Thus, according to a second analysis possibility, it is possible to normalise acquired signals as a function of a time elapsed between when the optical head passes in front of successive positioning marks associated with the scanned recognition areas.
According to a third possibility in which acquisition is also done without stopping, the relative displacement speed between the optical head and the chip can be servocontrolled continuously, by measuring the time elapsed between when the head passes in front of successive marks.
The invention also relates to a biochip that can be read by a device like that described above, in which the molecular recognition areas are superposed on the positioning marks in whole or in part, so as to cover them partially or completely.
This characteristic is particularly advantageous since it prevents congestion of the surface of the biochip by the positioning marks. It means that most or all of the entire surface of the chip can be left available for molecular recognition areas.
Positioning marks are used to orient the biochip, and also to mark and identify recognition areas. This is why they will preferably be located in predetermined positions with respect to the positioning marks.
Very thin positioning marks can be sufficient to position the biochip and/or identify recognition areas.
However, it is found that good precision is only achieved if the surface area of the positioning marks compared with the surface area of the molecular recognition areas with which they are associated, is not negligible.
The characteristic by which positioning marks are arranged so that they are partially or completely overlapped by the molecular recognition areas means that their area can be increased, and thus the servocontrol of position of the biochip relative to the read device can be improved. The read precision is also improved.
When positioning marks are in the form of guide tracks along which an incident light beam will travel for servocontrol of the position, it is desirable that the width of the track is not negligible compared with the width of the light beam.
The track can be marked with excellent precision using positioning marks with a surface that occupies of the order of 30 to 100% of the total area of the chip. overlapping of recognition areas and positioning marks appears even more economically attractive when considering the cost of manufacturing the biochips per unit useful area.
Furthermore, the increase in the area of the positioning marks makes it easier to read the marks and means that read devices can be used with a lower light power and with a lower sensitivity.
This also reduces the cost of the read devices.
The possibility of using a lower intensity light source for positioning reduces the risks of inhibiting or burning fluorescent markers that could be present on molecular recognition areas.
Furthermore, positioning marks may be designed to be almost transparent to at least one fluorescent light that could be emitted from recognition areas in response to at least one incident reading light.
When the laser beam reads through the back face, the reflectivity of the positioning marks and/or an interface between the coating material on recognition areas and the positioning marks may be chosen to be greater than 0%, preferably between 1 and 10%, and more precisely between 1 and 5%.
When the laser beam reads through the front face, the reflectivity of the positioning marks and/or an interface between the coating material on recognition areas and the positioning marks may be chosen to be greater than 0%, specifically between 1 and 100%.
The front and back faces of the biochip refer to the face coated with recognition molecules in the recognition areas (front face) and the opposite uncoated face (back face).
Furthermore, the biochip may comprise an intermediate layer made of a material with a reflectivity different from the reflectivity of the positioning marks, between the positioning marks and the recognition areas. The intermediate layer can then give a determined reflection of incident light on positioning markers, independently of the coating of the molecular recognition areas, and independently of the liquid or gas medium with which the biochip is brought into contact.
Other possibilities can be envisaged for making positioning marks. In particular, they may comprise a sequence of regions with different alternating properties for the incident light.
According to a first possibility, adjacent positioning mark regions may have a different optical path for incident light. The difference in the optical path may be obtained particularly by making adjacent regions from different materials, and/or with different thicknesses, and/or with different doping. The choice of different materials or different doping for adjacent regions means that the refraction indexes for each of them are different.
According to another possible means of making the positioning marks, these marks may be made of strips of bi-refringent material with the property of modifying the polarization direction of an incident polarized read light.
According to yet another possibility, adjacent positioning mark regions may be made from materials with different reflectivities.
Other characteristics and advantages of this invention will become clearer from the following description with reference to the figures in the attached drawings. This description is given purely for illustration and is in no way restrictive.